This Program Project contains four distinct projects with overlapping goals and methodology. Dr. Sulzer uses primary neuronal culture and a variety of biophysical methods including amperometry, electrophysiology and capacitance to assess the release of dopamine and its regulation by modulation of exocytosis, vesicle acidification and synaptic growth. The regulation of monoamine release and in particular vesicle filling originally led to the assembly of this group around a strong bond between the laboratories of Drs. Sulzer and Edwards. In addition, Drs. von Zastrow and Edwards have now extended their work in cell lines to transfection of primary hippocampal, striatal and cortical cultures, largely as a result of this Program Project, and interactions between themselves and Dr. Sulzer. The proximity of von Zastrow and Edwards labs has also encouraged an interest in the role of presynaptic receptors in the regulation of dopamine release, and its potential for plasticity in drug abuse. Since Dr. Kelly has assumed many administrative duties and accordingly reduced the size of his lab, he will no longer participate in this Program Project. In his place, we have added Dr. Ryan for his expertise with vesicle recycling. As a result of the methods they have used, Dr. Ryan has a very different perspective on transmitter release from Dr. Sulzer, and we expect that through the interaction supported by this Program, they will work together to resolve the very interesting conflicts. In addition, Dr. Ryan has a particular interest in dopamine neurons since it seems likely that they recycle synaptic vesicle membrane differently from other neuronal populations. Overall, the physiological perspective of Drs. Sulzer and Ryan will also help to guide the molecular analysis by Drs. Von Zastrow and Edwards. Conversely, Drs. Von Zastrow and Edwards will generate hypotheses about transmitter release and dopamine receptor regulation that Dr. Sulzer can test directly.